Nox1 generates ROS in physiological conditions and certain pathological conditions. ROS are known to contribute to damage within organisms and are thus associated with many different conditions. NADPH oxidases are also known as a source of oxidative stress, which contributes to the cause of many conditions. Nox1 expression is known to be associated with many different conditions, including cancers.
Therapeutic selectivity without toxicity is clearly important in a therapeutic agent. In particular preferential killing of cancer cells without toxicity to normal cells, is one of the most important considerations in cancer therapy.
Prior to the present invention, several inhibitors of NADPH oxidases (Nox) have been identified, but so far no specific Nox inhibitors have been developed.
Several cell-permeable peptide inhibitors, such as gp91ds-tat peptide, PR-39, as well as Rac peptide inhibitors have been also tested. The gp91ds-tat peptide was designed to inhibit specifically Nox2 by mimicking a sequence of Nox2 that is thought to be important for the interaction with p47phox. However, the peptide lacked specificity since the region targeted by the peptide is homologous in other NOX isoforms. In addition, the peptide had a low-efficacy inhibitor, inhibiting neutrophil ROS generation by 25% at 50 mM.
Recently, the company GenKyoTex developed more specific Nox inhibitors using Pyrazolo-pyrido-diazepine, -pyrazine and -oxazine dione derivatives, targeting in particular Nox1 and Nox4 enzymes. GenKyoTex is currently conducting a Phase I clinical with a small molecule, GKT137831 for the treatment of diabetic nephropathy. Preclinical experiments are also conducted for the treatment of diabetic nephropathy, atherosclerosis, idiopathic pulmonary fibrosis, liver fibrosis and models of angiogenesis. However, while being specific for Nox1/Nox4, GKT137831 also shows affinity for Nox2, Nox3, and Nox5, and thus has a low selectivity.
Other chemical compounds have been used for many years, including apocynin, diphenylene iodonium (DPI), and 4-(2-aminoethyl)-benzensulfonyl fluoride hydrochloride (AEBSF) and neopterin.
Apocynin, which was extracted from Picrorhiza kurroa, can prevent the formation of the active oxidase complex. The inhibitory effect of apocynin is however controversial. In fact, it was shown that apocynin is not specific for NADPH oxidases, but rather influences other events such as Thromboxane A2 formation and the induction of AP-1 transcription factor, and that it is an antioxidant, rather than a Nox enzyme inhibitor.
Diphenylene iodonium (DPI) is an unspecific inhibitor of all flavoenzymes, and thus can inhibit the Nox enzymes, but also xanthine oxidase and cytochrome P450 enzymes. In addition of being non-selective, DPI has been showed to be toxic. It is also an inhibitor of acetylcholinesterase and butyrylcholinesterase as well as of the internal Ca2+ pump, which in addition to its intrinsic toxicity raises major concerns on its application.
Aminoethyl-benzenesulfono-fluoride (AEBSF) has been shown to prevent the binding of flavocytochrome b558 to p47phox, the activation of the NADPH oxidase and the elicitation of O.2− production in macrophages. AEBSF is however primarily a serine protease inhibitor; it is thus capable of inhibiting with a higher efficiency proteases such as chymotrypsin, kallikrein; plasmin, thrombin, and trypsin, and has many additional effects.
Some other non-specific molecules, misleadingly named as indirect Nox inhibitors, have been shown to interfere with the upstream signal-transduction pathways affecting Nox enzymes. These include the VAS2870 which is a thiotriazolopyrimidine compound which inhibits PDGF-dependent Src activation of Nox enzymes, angiotensin converting enzyme inhibitors (ACE inhibitors), Ang II receptor blockers (ARBs), phosphodiesterase, eicosanoids, corticosteroids, MAP kinase, protein kinase C inhibitors (such as a benzo(b)pyran-4-one derivative, S17834 of Shionogi Pharma).
There are also an increasing number of reports using siRNA approaches directed against the Nox enzymes. Specifically some RNAi has been developed to knockdown Nox1 expression. However, the use of RNAi as a therapeutic intervention for humans is still in its infancy, and may take years to optimize. Unfortunately, only a few of the siRNAs have been properly tested for specificity of Nox isoforms. There are not any satisfactory siRNA approaches for selective inhibition of Nox, specifically Nox1.
Therefore the compounds which have been developed so far are not acting directly to block the enzyme, but either interfere with the upstream transduction pathway, or act as antioxidants or ROS scavengers. The only compounds that are capable of directly blocking the enzyme, however lack selectivity and inhibit other enzymatic activities. They also have low potency and bioavailability, thereby precluding a pharmacologic demonstration of Nox as therapeutic targets in vivo. Several small-molecule and peptide inhibitors of the Nox enzymes have been useful in experimental studies, but issues of specificity and toxicity militate against any of the existing compounds as candidates for drug development.
Given the broad array of disease targets documented in recent work, it was critical to find novel clinically useful inhibitors of the Nox enzymes. The greatest challenge was however the discovery of peptide inhibitors which are capable of acting specifically on individual Nox isoforms.
The Applicant has undertaken and successfully achieved this challenge. Indeed, the present invention provides novel peptide inhibitors that block directly and specifically NADPH oxidase-1 (Nox1) assembly, without inhibiting other NADPH noxidases, particularly Nox2, Nox4, Nox5, Duox1, and Duox2. Nox1 is involved in the development and progression of a wide spectrum of diseases, including cancer, aging, neurodegenerative diseases, and cardiovascular diseases, and thus represents a significant therapeutic target. The novel peptides according to the present invention are thus also particularly advantageous as they do not have non-specific ROS scavenging or antioxidant activity and they do not inhibit other sources of ROS. Compared to RNAi technology, the peptide inhibitors are very specific for their target protein, thereby reducing the likelihood of off-target effect. When compared to chemical inhibitors, the peptides according to the present invention showed no toxic effect in vivo. Furthermore, they are easy to design and produce.